Antigen-specific Abs are able to enhance or suppress immune responses depending on the receptors that they bind on immune cells. Recent studies have shown that pro- or antiinflammatory effector functions of IgG Abs are also regulated through their Fc N-linked glycosylation patterns. IgG Abs that are agalactosylated (non-galactosylated) and asialylated are proinflammatory and induced by the combination of T cell–dependent (TD) protein antigens and proinflammatory costimulation. Sialylated IgG Abs, which are immunosuppressive, and Tregs are produced in the presence of TD antigens under tolerance conditions. T cell–independent (TI) B cell activation via B cell receptor (BCR) crosslinking through polysaccharides or via BCR and TLR costimulation also induces IgG Abs, but the Fc glycosylation state of these Abs is unknown. We found in mouse experiments that TI immune responses induced suppressive sialylated IgGs, in contrast to TD proinflammatory Th1 and Th17 immune responses, which induced agalactosylated and asialylated IgGs. Transfer of low amounts of antigen-specific sialylated IgG Abs was sufficient to inhibit B cell activation and pathogenic immune reactions. These findings suggest an immune regulatory function for TI immune responses through the generation of immunosuppressive sialylated IgGs and may provide insight on the role of TI immune responses during infection, vaccination, and autoimmunity.

Abs regulate the production of new Abs specific for the same antigen via positive or negative feedback mechanisms (1–5). For example, IgG Abs form immune complexes (ICs) with the antigen and generate negative feedback regulation through crosslinking the B cell receptor (BCR) with the IgG inhibitory receptor FcγRIIB (encoded by Fcgr2b), which inhibits BCR signaling and thereby B cell activation and IgG Ab production (2, 3). In addition, FcγRIIB-independent negative feedback effects of IgG Abs have been described (4, 5). For example, T cell–independent type-2 (TI-2) antigen–induced IgG Abs suppress the B cell activation that results from a second challenge with the same TI-2 antigen, independent of FcγRIIB (5).

Recent studies have indicated that the activating or inhibitory functions of IgG Abs are also regulated through the pattern of the Fc glycan linked to Asn297 (6–16). The biantennary core glycan structure, which is composed of 2 N-acetyl-glucosamines (GlcNAc) and 3 mannoses, can be further modified with fucose, bisecting GlcNAc and terminal GlcNAc, galactose, and sialic acid.

The disease severity of RA has been associated with the appearance of proinflammatory asialylated (non-sialylated) and agalactosylated (G0) serum IgG auto-Abs (6, 11, 17–34). Furthermore, the anti-gp120 IgG Abs of HIV patients are less galactosylated and sialylated in long-term nonprogessors, who are infected but show no disease symptoms, compared with infected patients with disease symptoms (15). In contrast, IgG Abs that are both galactosylated and sialylated possess inhibitory properties that underlie the antiinflammatory effect of i.v. IgG (IVIG; pooled serum IgG from healthy donors), which is used in high doses (2 g/kg) to treat autoimmune patients (7–10, 12).

Regarding the physiological development and function of differentially glycosylated IgG Abs, it has recently been shown that tolerance induction with T cell–dependent (TD) protein antigen without a proinflammatory costimulus induces not only Tregs, but also immunosuppressive sialylated IgGs. Small doses (2.5 mg/kg) of sialylated antigen-specific IgGs, in the form of ICs, inhibit the maturation of DCs and proinflammatory immune responses in an antigen-specific manner (13). In contrast, the combination of TD antigens and TLR costimulation induces proinflammatory T and B cell immune responses, including the production of proinflammatory asialylated IgGs (13). For example, TD B cell activation via TLR/MyD88 costimulation plays a major role in the development of pathogenic IgG auto-Abs and autoimmunity, as demonstrated in different mouse models of lupus (35–41).

Recent evidence indicates that B cell activation via BCR and TLR costimulation without the help of T cells can also induce IgG Abs (40, 42–44), although the pro- or antiinflammatory Fc glycosylation pattern of TI-1 and TI-2 IgGs is unknown. In the present study, we compared TI-1 and TI-2 IgGs with respect to TD B cell activation and found that TI B cell activation was associated with the production of immunosuppressive sialylated serum IgGs, which inhibited B cell activation and immune reactions, independent of FcγRIIB.

To determine whether the suppressive effect of TI, CD154-independent antigen immunization could be overcome using adjuvant, we administered TNP-LPS in the presence of CFA or alum (Figure 1 and Supplemental Figures 1 and 2). However, neither CFA nor alum was sufficient to overcome the suppressive effect of TI TNP-LPS immunization on footpad swelling (Figure 1 and Supplemental Figure 2).

The sialic acid content of TNP-reactive IgGs induced with 50 μg TI TNP-LPS or TNP-Ficoll was similar to the steady-state level measured in purified total serum IgGs from nonimmunized WT and TCRβ/δ-deficient (Tcrbd–/–) mice as well as the level of TD OVA-reactive IgGs after tolerance induction with 2 mg OVA without costimulation (Figure 3B, Supplemental Figure 4, and ref. 13). The G0 content of the TI TNP-reactive IgGs was comparable to the levels in purified total serum IgGs from nonimmunized WT mice (Figure 3C). Similar results were obtained after TI antigen immunization in Fcgr2b–/– mice (Supplemental Figure 4, D–F).

In contrast, TNP- and OVA-reactive IgG Abs induced by TNP-OVA in CFA or by OVA in CFA showed significantly lower sialic acid contents than IgGs from untreated WT mice (13) or TI antigen–reactive IgG Abs. Only partial desialylation of TD anti-OVA IgGs was observed after Th2-mediated OVA with alum immunization (Figure 3B and Supplemental Figure 4). The G0 content after TD stimulation with OVA was higher than the total IgG content from untreated mice and that of TI TNP-reactive IgGs (Figure 3C). Non–antigen-reactive serum IgGs at the same time point after immunization showed galactosylation and sialylation levels comparable to the total serum IgG obtained from untreated mice (data not shown).

However, TI TNP-LPS immunization with CFA costimulation was not sufficient to induce low-sialylated and low-galactosylated anti-TNP IgGs (Figure 3, B and C). These results indicated that T cell help is important for the induction of asialylated and agalactosylated antigen-reactive IgGs under proinflammatory conditions. Accordingly, low sialylation levels of OVA-reactive serum IgGs induced by OVA in CFA were dependent on the synergistic effects of the Th1 cytokine IFN-γ and the Th17 cytokine IL-17, as demonstrated by the partial lack of proinflammatory low-sialylated anti-OVA IgGs in Ifngr1–/– and Il17ra–/– mice and their complete absence in double-deficient Ifngr1–/–Il17ra–/– mice (Figure 3D and Supplemental Figure 5). The increased G0 content of OVA-reactive IgGs after OVA in CFA immunization was dependent on IFN-γRI signaling (Figure 3E).

In summary, these findings showed that TD protein antigens in the context of a proinflammatory Th1 and Th17 cell–inducing costimulus induced proinflammatory agalactosylated and asialylated IgG Abs, whereas even this costimulus was not sufficient to induce agalactosylated and asialylated IgG Abs after TI immunization. Thus, TI IgG Abs are galactosylated and sialylated.

Antigen-specific sialylated, but not desialylated, IgG Abs suppress pathogenic immune reactions and B cell activation, independent of FcγRIIB. To determine whether the suppressive function of TI IgGs is directly associated with their sialylation level, we transferred native sialylated or sialidase-treated desialylated TNP-reactive serum IgGs (purified from TNP-LPS–immunized mice) into recipient mice 1 day before inducing DTH with TNP-OVA in alum (Figure 4, A and B). In addition, to investigate whether the suppressive function of sialylated IgGs is independent of FcγRIIB, we used Fcgr2b–/– mice as recipients. Transfer of 200 μg of native sialylated, but not sialidase-treated desialylated, TI TNP-specific serum IgGs was sufficient to reduce the pathogenic DTH response, as measured by footpad swelling, anti-OVA IgG2b serum Ab levels, and CD4+ T cell accumulation in local LNs (Figure 4, C–E).

We further found that just 100 μg of 15% sialylated anti-TNP murine IgG1 (clone H5) was sufficient to reduce a DTH response in both WT and Fcgr2b–/– mice in an antigen-specific manner (Supplemental Figure 6). Sialylation of clone H5 IgG1 Abs did not influence TNP binding (Supplemental Figure 6 and ref. 13).

TI-2 antigens have been implicated in the induction of suppressive IgGs, which inhibit a secondary B cell challenge with the same TI-2 antigen, independent of FcγRIIB (5). Furthermore, it has been shown that IVIG treatment can inhibit B cell activation, independent of FcγRIIB (55, 56). To further examine the influence of sialylated IgGs on B cell activation, B cells from WT and Fcgr2b–/– mice were stimulated in vitro with LPS and treated with native asialylated (<1% sialylation) or in vitro sialylated (15% sialylation) monoclonal anti-TNP murine IgG1 hybridoma Abs (clone H5; Figure 5, Supplemental Figure 6, and ref. 13). Asialylated monoclonal murine IgG1 Abs reduced the proliferation of WT B cells, but not Fcgr2b–/– B cells (Figure 5), obviously through an FcγRIIB-dependent mechanism (4, 54). However, sialylated IgG1 Abs inhibited the proliferation of both WT and Fcgr2b–/– B cells (Figure 5), through an FcγRIIB-independent mechanism.

With respect to the results shown in Figures 1 and 2, IgM Abs can also have immunoregulatory properties (1, 41, 49–53). To analyze the feedback effect of differentially sialylated IgM Abs on TI antigen–induced B cell activation, a native sialylated or sialidase-treated desialylated monoclonal anti-TNP IgM Ab was complexed with TNP-Ficoll and injected i.p. into WT mice (Supplemental Figures 7 and 8). The desialylated, but not sialylated, monoclonal IgM Ab enhanced the TNP-Ficoll–induced anti-TNP IgG response. Although the sialylated monoclonal IgM Ab failed to reduce the TNP-Ficoll–induced anti-TNP IgG response in this experiment, these data demonstrated that the sialylation of IgM Abs influences their feedback effect.

In summary, our data showed that TI B cell activation leads to the development of immunosuppressive sialylated IgG Abs, which can inhibit immune responses, such as B cell activation, independent of FcγRIIB.

The induction of either a proinflammatory immune response or tolerance to an antigen is a major challenge for the immune system. TD protein antigens in the context of proinflammatory costimuli induce proinflammatory T cells and IgG Abs with low Fc sialylation, whereas TD immune responses without costimulation induce peripheral T cell tolerance (57) and immunosuppressive sialylated IgG Abs (13). Our present results showed that TI-1 and TI-2 B cell activation also induced immunosuppressive sialylated IgG Abs, even in the presence of proinflammatory costimuli. Thus, the help of T cells (especially Th1 and Th17 cells), together with proinflammatory costimuli, are important for the induction of proinflammatory IgG immune responses. In contrast, TD protein antigens under tolerogenic conditions and TI antigens induced sialylated immunosuppressive IgG Abs.

Strategies to improve the success of vaccines have shown that TI antigens, such as polysaccharides, should be coupled to a foreign protein to recruit T cell help (58). The present observation that TI immunizations alone induced suppressive sialylated IgGs may explain this positive protein effect in vaccines. Furthermore, IgG Fc asialylation was more dependent on Th1 and Th17 than on Th2 responses, which may also be important for adjuvant selection in vaccine development.

Different murine models for autoimmune lupus have demonstrated that Th1 and Th17 cells and TD B cell activation via TLR/MyD88 costimulation play a major role in the development of highly mutated, high-affinity pathogenic IgG auto-Abs and autoimmune disease. Recent studies have also shown that TI-1 B cell activation via BCR and TLR costimulation is sufficient to induce IgG auto-Abs (35–45). However, whether the presence of IgG auto-Abs generated in response to TI-1 B cell activation via TLR7 or TLR9 costimulation reflects inflammatory lupus disease or protection must be investigated, as well as the Fc glycosylation pattern of the TI IgG auto-Abs.

The data obtained in the present study showed that TI B cell activation leads to the development of suppressive sialylated IgGs similar to the sialylation level of total serum IgG under steady-state conditions in nonimmunized mice. Steady-state IgG contains both autoreactive and polyreactive IgG Abs, which may play an important role in maintaining self-tolerance (59, 60). TI B cell activation also induces the production of IgM Abs, which may also have suppressive feedback effects, as was previously suggested for IgM Abs in a different context (1, 41, 49–53). In the present study, a desialylated monoclonal anti-TNP IgM Ab enhanced TNP-Ficoll–induced B cell activation in vivo. However, the sialylated anti-TNP IgM Ab failed to inhibit the TI response. The anti-TNP IgM Ab used in this study was a monoclonal Ab produced in HEK293 cells. Sialylated polyclonal serum IgM Abs might have other characteristics that mediate suppressive effects, including different levels of high-mannose and hybrid structures (Supplemental Figure 8) and the presence of the J-chain with unclear functions. However, our data showed that sialylation at least inhibited the positive feedback effects of IgM Abs on TI antigen–induced B cell activation.

The inhibitory properties of sialylated IgGs have been previously described and underlie the antiinflammatory effect of IVIG (7, 8, 10, 12). The sialylated subfraction of IVIG actively modulates marginal zone macrophages via the C-type lectin receptor specific ICAM-3 grabbing nonintegrin–related 1 (SIGN-R1), which is primarily independent of FcγRIIB (8, 12). Sialylated IgGs have a 10-fold lower affinity for activating and inhibiting FcγRs than do asialylated IgGs (7), which further suggests that mechanisms and/or receptors other than FcγRIIB mediate the suppressive effect of sialylated IgGs. Accordingly, small amounts of ICs containing sialylated IgGs were shown to inhibit the maturation of DCs and proinflammatory immune responses in an antigen-specific manner, independent of FcγRIIB (13).

In summary, our findings identified a novel immune regulatory function for TI immune responses through the generation of antigen-specific immunosuppressive sialylated IgG Abs, which further indicates that the presence of antigen-specific IgG serum Abs is not sufficient to predict the inflammatory quality of an overall immune response. Furthermore, these data may have important implications for understanding TI immune responses to self-antigens and pathogens and for the development of novel vaccination strategies using TI antigens.

Immunization and anti-OVA and anti-TNP IgG Ab purification. 8-week-old mice were immunized i.p. as indicated in Figure 3 and Supplemental Figures 4 and 5. Serum samples were collected on day 14, and pooled serum IgGs were purified with protein G–sepharose. OVA- or TNP-reactive IgGs were additionally purified using OVA or TNP-BSA coupled to a CNBr-activated sepharose 4B column (GE Healthcare) prepared in our laboratory. Antigen-reactive IgG reached up to 5% of total serum IgG, depending on the stimulus (data not shown). Enrichment of OVA- or TNP-reactive IgGs was verified via ELISA. IgG Fc glycan structures were analyzed by MALDI-TOF mass spectrometry (MS).

In vitro sialylation and desialylation of IgG and IgM Abs. Murine anti-TNP IgG1 hybridoma cells (clone H5) (63) and murine anti-Thy1.1 IgG1 hybridoma cells (clone MRC OX-7) were grown for Ab production in 0.03% Primatone RL/UF. IgG Abs were purified from cell culture media with protein G–sepharose. In vitro sialylation of purified IgG Abs was performed in a 2-step procedure as described previously (7, 13). Briefly, Abs were galactosylated with human β1,4-galactosyltransferase and UDP-galactose and subsequently sialylated with human α2,6-sialyltransferase and CMP–sialic acid (all from Calbiochem). Desialylation of TNP-reactive IgG Abs purified from the pooled sera of TNP-LPS–immunized mice or recombinant monoclonal anti-TNP IgM Abs was performed using a Prozyme Sialidase kit (no. GK80040).

Glycan analysis. Total or antigen-specific IgG samples were digested with recombinantly expressed endoglycosidase S (EndoS) from Streptococcus pyogenes (64). EndoS specifically cleaves the N-linked glycan at the Fc fragment between the first and second GlcNAc (Figure 3A, Supplemental Figure 4, and ref. 64). Monoclonal anti-TNP IgM Abs were digested with PNGaseF (Supplemental Figure 8 and ref. 65). The resulting N-glycans were purified by solid-phase extraction using reversed-phase C18 and graphitized carbon columns (Alltech). The samples were permethylated according to standard protocols (66) and further investigated by MALDI-TOF MS in duplicate. The spectra were recorded on an Ultraflex III mass spectrometer (Bruker Daltonics) equipped with a Smartbeam laser. Calibration was performed on a glucose ladder, and 2,5-dihydroxybenzoic acid was used as matrix. The spectra were recorded in reflector positive ionization mode, and mass spectra from 3,000 laser shots were accumulated.

DTH responses. DTH responses were induced by immunization with 100 μg TNP-OVA in alum on day 0, followed by injection with 37.5 μg OVA in montanide ISA 50V (Seppic) into the right footpad on the indicated day. PBS in montanide was injected into the left footpad as a negative control. The difference in footpad thickness between the right and left footpad was determined using an Oditest micrometer gauge (Kroeplin) in a blinded manner.

In vitro B cell proliferation. A total of 4 × 105 spleen cells was cultured in RPMI medium containing 10% FCS without IgG (prepared in our laboratory) and treated with 10 μg/ml LPS (Sigma-Aldrich) in 96-well plates. In addition, the cells were incubated with native <1% or 15% sialylated anti-TNP murine IgG1 (clone H5; Supplemental Figure 6). On day 3, total cell numbers were counted and analyzed by FACS after staining for B220 and DAPI to calculate the total numbers of live B cells.

Statistics. Statistical analyses were performed using 2-tailed Student’s t test or the log-rank test for survival curves. A P value less than 0.05 was considered significant.

Study approval. Mice were bred, maintained, and used in experiments in compliance with approved local institutional animal care and use regulations and with permission of the regional authorities in Berlin, Germany.

We thank Andreas Radbruch and Fritz Melchers for discussion; Angelina Jahn, Heidi Hecker-Kia, Heidi Schliemann, Tuula Geske, Toralf Kaiser, Katharina Raba, and Detlef Grunow for technical assistance; Mattias Collin for support with EndoS; Birgitta Heyman for support with the anti-TNP IgG1 H5 hybridoma; and J. Tocker for providing Il17ra–/– mice. M. Ehlers was a fellow of the Claussen-Simon-Foundation and was supported by the DFG (EH221-4, EH221-5, RTG 1727, IRTG 1911, SFB/TR 654, and the Excellence Cluster ‘‘Inflammation at Interfaces”) and by the Max Planck Institute for Infection Biology. D. Petzold was supported by the RTG 1727 “Modulation of Autoimmunity” (DFG). S. Eiglmeier was supported by the International Max Planck Research School for Infectious Diseases and Immunology. V. Blanchard and M. Berger were supported by the German Ministry of Research and Education (03IP511) and the Sonnefeld Foundation.

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